The invention relates to novel adenylate cyclase nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
Adenylate cyclase is a membrane-bound enzyme that acts as an effector protein in a receptor-effector system referred to as the cAMP signal transduction pathway. As such, it plays a key intermediate role in the conversion of extracellular signals, perceived by various receptors following binding of a particular ligand, into intracellular signals that, in turn, generate specific cellular responses.
A variety of hormones, neurotransmitters, and olfactants regulate the synthesis of cAMP by adenylate cyclases. In most tissues, regulation of cAMP synthesis is accomplished through three plasma membrane-associated components: G-protein-coupled receptors (GPCRs), which interact with regulatory hormones and neurotransmitters; heterotrimeric G proteins that either stimulate or inhibit the catalytic subunit of adenylate cyclase in response to interaction of ligands with appropriate GPCRs; and the catalytic entity, adenylate cyclase. Each G protein contains a guanine nucleotide-binding alpha subunit and a complex of tightly associated xcex2- and xcex3-subunits. When a G protein is activated following binding of a ligand to a GPCR, GDP is released from the xcex1-subunit in exchange for GTP. Binding of the GTP results in conformational changes that yield dissociation of the GTP-bound a-subunit from the xcex2-xcex3-subunit complex. The resulting macromolecular complexes regulate catalytic activity of adenylate cyclase. Where the receptor is a stimulatory receptor (Rs), interaction with a stimulatory G-protein, termed Gs, results in activation of the adenylate cyclase catalytic subunit by the GTP-bound form of the Gs xcex1-subunit. In contrast, where the receptor is an inhibitory receptor (Ri), interaction with an inhibitory G-protein (one of several known Gis) results in inhibition of the adenylate cyclase catalytic subunit by the GTP-bound form of the Gi xcex1-subunit. In addition, the G-protein xcex2-xcex3-subunit complex may interact with and influence adenylate cyclase activity independent of or in parallel with the GTP-bound xcex1-subunit, depending upon the adenylate cyclase isoform involved. See Taussig and Gilman (1995) J. Biol. Chem. 6:1-4; Hardman et al., eds. (1996) Goodman and Gilman""s Pharmacological Basis of Therapeutics (McGraw-Hill Company, New York, N.Y.).
When activated, the catalytic subunit of adenylate cyclase converts intracellular ATP into cAMP. This second messenger then activates protein kinases, particularly protein kinase A. Activation of this protein kinase causes the phosphorylation of downstream target proteins involved in a number of metabolic pathways, thus initiating a signal transduction cascade.
The extent to which adenylate cyclase converts ATP to cAMP is highly dependent on the state of phosphorylation of the various components of the hormone-sensitive adenylate cyclase system. For example, stimulatory and inhibitory receptors are desensitized and down-regulated following phosphorylation by various kinases, particularly cAMP-dependent protein kinases, protein kinase C, and other receptor-specific kinases that preferentially use agonist-bound forms of receptors as substrates. In this manner, tight regulation of the cellular cAMP concentration, and hence regulation of the cAMP signal transduction pathway, is achieved (Taussig and Gilman (1995) J. Biol. Chem. 270:1-4).
Adenylate cyclase activation may also occur through increased intracellular calcium concentration, especially in nervous system and cardiovascular tissues. After depolarization, the influx of calcium elicits the activation of calmodulin, an intracellular calcium-binding protein. In the cardiovascular system, this effect gives rise to the contraction of the blood vessels or cardiac myocytes. The activated calmodulin has been shown to bind and activate some isoforms of adenylate cyclase.
Several novel isoforms of mammalian adenylate cyclase have been identified through molecular cloning. Type I adenylate cyclase (CYA1) is primarily localized in brain tissues (see Krupinski et al (1989) Science 244:1558-1564; Gilman (1987) Ann. Rev. Biochem. 56:615-649, citing Salter et al. (1981) J. Biol. Chem. 256:9830-9833; Andreasen et al. (1983) Biochemistry 22:2757-2762; and Smigel et al (1986) J. Biol Chem. 261:1976-1982 for bovine CYA1; and Villacres et al. (1993) Genomics 16:473-478 for human CYA1). The type II adenylate cyclase (CYA2) is localized in brain and lung tissues (see Feinstein et al. (1991) Proc. Natl. Acad. Sci. USA 88:10173-10177 for rat CYA2; and Stengel et al. (1992) Hum. Genet. 90:126-130 for human CYA2). Type III adenylate cyclase (CYA3) is primarily localized in olfactory neuroepithelium and is thought to mediate olfactory receptor responses (Bakalyar and Reed (1990) Science 250:1403-1406; Glatt and Snyder (1993) Nature 361:536-538; and Xia (1992) Neurosci. Lett. 144:169-173). Type IV adenylate cyclase (CYA4) most resembles type II, but is expressed in a variety of peripheral tissues and in the central nervous system (Gao and Gilman (1991) Proc. Natl. Acad. Sci. USA 88:10178-10182, for rat CYA4). Type V adenylate cyclase (CYA5) (Ishikawa et al. (1992) J. Biol. Chem. 267:13553-13557; Premont et al. (1992) Proc. Natl. Acad. Sci. USA 89:9809-9813; and Glatt and Snyder (1993) Nature 361:536-538; Krupinski et al. (1992) J. Biol. Chem. 267:24858-24862) and type VI adenylate cyclase (CYA6) (Premont et al. (1992) Proc. Natl. Acad. Sci. USA 89:9808-9813; Yoshimura and Cooper (1992) Proc. Natl. Acad. Sci. USA 89:6716-6720; Katsushika et al. (1992) Proc. Natl. Acad. Sci. USA 89:8774-8778; and Krupinski etal. (1992) J. Biol. Chem. 267:24858-24862) both exhibit a widely distributed expression pattern, with type V having high expression in heart and striatum, and type VI having high expression in heart and brain. Type VII adenylate cyclase (CYA7) is widely distributed, though may be absent from brain tissues (Krupinski et al (1992) J. Biol. Chem. 267:24858-24862). Type VIII adenylate cyclase (CYA8) is abundant in brain tissues (Krupinski et al. (1992) J. Biol. Chem. 267:24858-24862; and Parma et al. (1991) Biochem. Biophys. Res. Commun. 179:455-462). Type IX adenylate cyclase (CYA9) is widely expressed, at high levels in skeletal muscle and brain (Premont et al. (1996) J. Biol. Chem. 271:13900-13907).
The different isoforms of adenylate cyclase exhibit unique patterns of regulatory responses (see Sunahara et al. (1996) Annu. Tev. Pharmacol. Toxicol 36:461-480). For example, all of these isoforms are activated by the xcex1-subunit of a particular G protein, termed Gs, which couples the stimulatory action of the ligand-bound receptor to activation of adenylate cyclase. The adenylate cyclases designated type I, III, and VIII are also stimulated by Ca2+/calmodulin in vitro, while type II, IV, V, VI, VII, and IX are not. Type I is inhibited by G protein xcex2-xcex3-subunit complex, independently of Gs activation, while Type II is highly stimulated by G protein xcex2-xcex3-subunit complex when simultaneously activated by Gs alpha subunit. Type III, in contrast, is not affected by G protein xcex2-xcex3-subunit complex. Type V and type VI are both are inhibited by low levels of Ca2+, but appear to be unaffected by G protein xcex2-xcex3-subunit complex. Type IX is unique in that it is stimulated by Mg2+, but is not affected by G protein xcex2-xcex3-subunit complex.
The genes for these adenylate cyclases all encode proteins having molecular weights of approximately 120,000 and which range from 1064 to 1353 amino acid residues. These proteins are predicted to have a short cytoplasmic amino terminus followed by a first motif consisting of six transmembrane spans and a cytoplasmic (domain C1), and then a second motif, also consisting of six transmembrane spans and a second cytoplasmic domain (domain C2). The two cytoplasmic domains are approximately 40 kDa each and contain a region of homology (designated C1a and C2a) with each other and with the catalytic domains of membrane-bound guanylate cyclases. Based on this similarity, these domains are considered to be nucleotide binding domains, and together have been shown to be sufficient to confer enzymatic activity (Tang and Gilman (1995) Science 268:1769-1772).
Alterations in the cAMP signal transduction pathway have been associated with diseases such as asthma, cancer, inflammation, hypertension, atherosclerosis, heart failure. Antihypertensive drug therapy involves modulation of adenylate cyclase levels (Marcil et al. (1996) Hypertension 28:83-90). In addition, studies of heart in human and animal models indicate that adenylate cyclase has a function in cardiomyopathy (Michael et al. (1995) Hypertension 25:962-970, Roth et al. (1999) Circulation 99:3099-3102), ischemia (Sandhu et al. (1996) Circulation Research 78:137-147), myocardial infarction (Espinasse et al. (1999) Cardiovascular Research 42:87-98) and congestive heart failure (Kawahira et al. (1998) Circulation 98:262-267, Panza et al. (1995) Circulation 91:1732-1738). The enzyme is also related to some mental disorders. Studies of learning and memory in animal models indicate a likely role for calmodulin-activated adenylate cyclases in conditioning (Abrams and Kandel (1988) Trends Neurosci. 11: 128-135), learning (Livingstone et al. (1984) Cell 37:205-215), and long term potentiation (Frey et al. (1993) Science 260:161-1664). Furthermore, the cAMP signaling pathway plays an important role in cardiovascular physiology. For instance, cAMP activates protein kinase A (PKA). The activated subunits of PKA initiate a series of enzymatic reactions that ultimately activate multiple proteins that regulate both the rate and force of cardiac contraction.
Given the key role of adenylate cyclase in cAMP production, novel adenylate kinase molecules for the modulation of the cAMP signal transduction pathway are needed.
Isolated nucleic acid molecules corresponding to adenylate cyclase nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:2 or the nucleotide sequences encoding the DNA sequence deposited in a bacterial host with the Patent Depository of the American Type Culture Collection (ATCC) as Patent Deposit Number 1661. Further provided are adenylate cyclase polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptides or peptides of the invention by recombinant techniques.
The adenylate cyclase molecules of the present invention are useful for modulating cellular growth and/or cellular metabolic pathways, particularly for regulating the cAMP signal transduction pathway and phosphorylation of proteins via cAMP-dependent protein kinases involved in cellular growth and metabolism. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding adenylate cyclase proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of adenylate cyclase-encoding nucleic acids.
Another aspect of this invention features isolated or recombinant adenylate cyclase proteins and polypeptides. Preferred adenylate cyclase proteins and polypeptides possess at least one biological activity possessed by naturally occurring adenylate cyclase proteins.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequences set forth in the sequence listings are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid sequences are provided.
Antibodies and antibody fragments that selectively bind the adenylate cyclase polypeptides and fragments are provided. Such antibodies are useful in detecting the adenylate cyclase polypeptides as well as in regulating the T-cell immune response and cellular activity, particularly growth and proliferation.
In another aspect, the present invention provides a method for detecting the presence of adenylate cyclase activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of adenylate cyclase activity such that the presence of adenylate cyclase activity is detected in the biological sample.
In yet another aspect, the invention provides a method for modulating adenylate cyclase activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) adenylate cyclase activity or expression such that adenylate cyclase activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to adenylate cyclase protein. In another embodiment, the agent modulates expression of adenylate cyclase protein by modulating transcription of an adenylate cyclase gene, splicing of an adenylate cyclase mRNA, or translation of an adenylate cyclase mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the adenylate cyclase mRNA or the adenylate cyclase gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant adenylate cyclase protein activity or nucleic acid expression by administering an agent that is an adenylate cyclase modulator to the subject. In one embodiment, the adenylate cyclase modulator is an adenylate cyclase protein. In another embodiment, the adenylate cyclase modulator is an adenylate cyclase nucleic acid molecule. In other embodiments, the adenylate cyclase modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (1) aberrant modification or mutation of a gene encoding an adenylate cyclase protein; (2) misregulation of a gene encoding an adenylate cyclase protein; and (3) aberrant post-translational modification of an adenylate cyclase protein, wherein a wild-type form of the gene encodes a protein with an adenylate cyclase activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of an adenylate cyclase protein. In general, such methods entail measuring a biological activity of an adenylate cyclase protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the adenylate cyclase protein.
The invention also features methods for identifying a compound that modulates the expression of adenylate cyclase genes by measuring the expression of the adenylate cyclase sequences in the presence and absence of the compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.